Substituted oximes are useful pharmaceutical compounds. For example, International Application No. PCT/US98/23255, filed Nov. 18, 1998, describes certain substituted oximes that are useful as neurokinin antagonists. In many instances, it is desireable to produce a particular enantiomer of the substituted oxime. Thus, any efficient enantioselective process for producing a chiral intermediate for these compounds would be a welcome contribution to the art. This invention provides such a contribution.
This invention provides a process for preparing an S-enantiomer compound having the formula 
in enantiomeric excess, or an R-enantiomer compound having the formula 
in enantiomeric excess, said process comprising hydrolyzing a compound having the formula 
with:
(a) an enzyme capable of producing an enantiomeric excess of the S-enantiomer compound of formula (IA) of at least 70%, or
(b) an enzyme capable of producing an enantiomeric excess of the R-enantiomer compound of formula (IB) of at least 70%, wherein R1 is selected from the group consisting of alkyl, aryl, aralkyl, cycloalkyl, or cycloalkylalkyl.
As used herein, the term xe2x80x9calkylxe2x80x9d means straight or branched hydrocarbon chains of 1 to 6 carbon atoms, optionally substituted with one or more halo, hydroxy, or alkoxy substituents.
xe2x80x9cAlkoxyxe2x80x9d refers to a group having the formula Rxe2x80x94Oxe2x80x94, wherein R is alkyl.
xe2x80x9cArylxe2x80x9d refers to a carbocyclic group having at least one aromatic ring (e.g., phenyl or naphthyl), optionally substituted with one or more substituents selected from halo, alkyl, hydroxy, alkoxy, or xe2x80x94CF3.
xe2x80x9cAralkylxe2x80x9d refers to a group having the formula aryl-Rxe2x80x94, wherein R is alkyl.
xe2x80x9cCycloalkylxe2x80x9d refers to a non-aromatic carbocyclic ring of from 3 to 6 carbon atoms, optionally substituted with one or more substituents selected from halo, alkyl, hydroxy, alkoxy, or xe2x80x94CF3.
xe2x80x9cCycloalkylalkylxe2x80x9d refers to a group having the formula cycloalkyl-Rxe2x80x94, wherein R is alkyl.
xe2x80x9cHaloxe2x80x9d refers to fluorine, chlorine, bromine or iodine.
xe2x80x9cEtxe2x80x9d refers to an ethyl group.
xe2x80x9cEnantiomeric excessxe2x80x9d is calculated according to the following formula:       e    .    e    .          xe2x80x83        ⁢    %    =                    "LeftBracketingBar"                              [            R            ]                    -                      [            S            ]                          "RightBracketingBar"                              [          R          ]                +                  [          S          ]                      xc3x97    100    ⁢    %  
where [R] is the concentration of the R-enantiomer, and [S] is the concentration of the S-enantiomer.
R1 is preferably alkyl, more preferably methyl or ethyl, most preferably ethyl.
Enzymes suitable for use in the present process can be identified by carrying out the screening procedure described in Example 1, below. The enzyme is preferably one that is capable of producing an e.e. of the desired compound of at least 80%, more preferably at least 90%. Preferably, the enzyme is one that produces the S-enantiomer compound in enantiomeric excess.
Preferably, the enzymes used for preparing the S-enantiomer compound of formula (IA) are produced by Candida rugosa, Candida cylindracea, Candida antarctica, and Rhizopus delemar. Examples of enzymes suitable for use in preparing the S-enantiomer compound of formula (IA) include, but are not limited to, the following commercially available enzyme preparations: Altus ChiroCLEC CRO (Candida rugosa); Altus ChiroCLEC-CR (Candida rugosa); Altus Lipase CR Analytical Grade 001-C (Candida rugosa); Biocatalysts Ltd. (Candida cylindracea); Meito Sangyo LIPASE-OF (Candida cylindracea); Boehringer Mannheim Cholesterol Esterase (Candida rugosa); Boehringer Mannheim CHIRAZYME L-2 (Candida antarctica, fraction B); Fluka Lipase (Candida antarctica); Genzyme Lipase (Candida cyclindracea); Novo Nordisk Novozym 435 (Candida antarctica, type B); Novo Nordisk SP 525 (Candida antarctica, type B); and Seikagaki Lipase (Rhizopus delemar). Of these commercially available enzyme preparations, Boehringer Mannheim CHIRAZYME L-2, in either the dry or liquid form, and Novo Nordisk Novozym 435 (Candida antarctica, type B), in either the dry or liquid form, are particularly preferred.
Preferably, the enzymes used for preparing the R-enantiomer compound of formula (IB) are obtained from porcine or bovine pancreas. Examples of enzymes suitable for use in preparing the R-enantiomer compound of formula (IB) include, but are not limited to, the following commercially available enzyme preparations: Biocatalysts Ltd. Lipase (porcine pancreas); Boehringer Mannheim Lipase (porcine pancreas); Boehringer Mannheim CHIRAZYME L-7 Lipase (porcine pancreas); Rohm Tech COROLASE PP (porcine pancreas); Scientific Protein Labs. PEC High Lipase; Sigma xcex1-Chymotrypsin Type II (bovine pancreas); Sigma Trypsin (porcine pancreas); Sigma Lipase Type II (porcine pancreas); ThermoGen ThermoCat E001; ThermoGen ThermoCat E002; ThermoGen ThermoCat E003; ThermoGen ThermoCat E004; ThermoGen ThermoCat E005; ThermoGen ThermoCat E006; ThermoGen ThermoCat E007; ThermoGen ThermoCat E008; ThermoGen ThermoCat E009; ThermoGen ThermoCat E010; ThermoGen ThermoCat E011; ThermoGen ThermoCat E012; ThermoGen ThermoCat E013; ThermoGen TherrnoCat E014; ThermoGen ThermoCat E015; ThermoGen ThermoCat E016; ThermoGen ThermoCat E017B; ThermoGen ThermoCat 018; ThermoGen ThermoCat 019; ThermoGen ThermoCat 020; and ThermoGen ThermoCat 027. Of these commercially available enzyme preparations, Sigma xcex1-Chymotrypsin Type II and Boehringer Mannheim CHIRAZYME L-7 are particularly preferred.
The hydrolysis of compound (II) is preferably carried out at a pH of 5-9, more preferably 6-8.5, most preferably 7-8. Preferably, the substrate concentration is 5% to 25%, more preferably 8% to 12%. Preferably, the hydrolysis is carried out at a temperature of 25xc2x0 to 45xc2x0 C., more preferably, 30xc2x0 to 40xc2x0 C. The hydrolysis is preferably carried out in the presence of a buffer (e.g., sodium phosphate or potassium phosphate buffer), and the reaction may, if desired, be titrated with a caustic titrant (e.g., NaOH solution) to maintain the pH within the preferred range. Preferably, the concentration of the caustic titrant is 0.2 to 1 M, preferably about 0.5 M. In a particularly preferred embodiment, compound (II) is dispersed in a buffer solution by agitation, and the enzyme is subsequently added to the mixture. Upon adding the enzyme, the reaction mixture is agitated until the desired degree of hydrolysis is reached. When using an enzyme in solid form (e.g., adsorbed on beads), the reaction may be terminated by removing the enzyme by filtration, and adding acid (e.g., H2SO4) to the filtrate to adjust the pH to about 4.0-4.5, thereby precipitating the desired product. When using a liquid enzyme, the reaction may be terminated by adding acid (e.g., H2SO4) to adjust the pH to about 4.0-4.5, and the precipitated product can be recovered by filtration.
Compound (II) may be made by conventional means, e.g., as shown in the scheme below: 
As shown in the scheme above, aldehyde (1) is condensed with ethyl acetoacetate in ethyl alcohol with a piperidine catalyst to form crude reaction product (2), a mixture of 2 isomers. The condensation is preferably carried out over 2-3 days at 20xc2x0 C.xc2x15xc2x0 C. Potassium hydroxide is added to the crude reaction product (2), and heated at about 60xc2x0-70xc2x0 C. for about one hour to form dipotassium salt (3), which is filtered and washed with ethyl alcohol. The dipotassium salt is dissolved in water and treated with HCl to form compound (4). Compound (4) is reacted with an alcohol, R1OH (e.g., ethyl alcohol) in the presence of p-toluenesulfonic acid to form compound (II). Compound (II) may be isolated and purified by adding a higher boiling solvent, (e.g., heptane), distilling off the alcohol, washing with dilute sodium bicarbonate solution to remove the p-toluenesulfonic acid, and cooling the solution to precipitate compound (II).